Method and kit for non-invasively detecting egfr gene mutations

ABSTRACT

The present invention discloses a method for non-invasively detecting EGFR gene mutations in subjects, comprising the following steps: designing primers according to EGFR gene exons; extracting plasma DNAs in subjects; connecting the extracted plasma DNAs with tagging linkers; PCR pre-amplifying the tagging linkers connected plasma DNAs; cyclising the pre-amplified DNAs to obtain cyclised DNAs; PCR amplifying the cyclised DNAs using the designed primers; and high throughput sequencing the PCR amplified product and analyzing the EGFR gene mutations. The present invention also discloses a corresponding kit.

FIELD OF INVENTION

The present invention relates to genetic diagnosis field. Morespecifically, the present invention is directed to a method fordetecting Epidermal Growth Factor Receptor (EGFR) gene mutations, aswell as a kit for use in the detection of EGFR gene mutations.

BACKGROUND OF INVENTION

EGFR (Epidermal Growth Factor Receptor) is normally embedded in the cellmembrane on the cell surface. EGFR gene is 118 kb in length, includes 28exons, and encodes a 170 kD glycoprotein comprised of 1186 amino acids.It is a membrane protein that plays an important role in regulatingproliferation, growth, repair and survival of tumor cells. Currently,targeted therapy has become an important tool in the clinical treatmentof Non Small Cell Lung Cancer (NSCLC). Iressa (Gefitinib, AstraZeneca)and Tarceva (Erlotinib, Roche), which function as EGFR tyrosine kinaseinhibitors (TKI), are the main drugs approved by FDA for NSCLC targetedtherapy. However, clinical experiments showed that Iressa and Tarcevaonly had significant therapeutic effects on 10-30% NSCLC patients.Further studies indicated that EGFR gene mutations relate to thetherapeutic effects of NSCLC targeted therapy, and most of the patientscarrying EGFR gene mutations showed significant therapeutic effects. Alarge number of research documents indicated that EGFR gene mutationsare mainly located in the tyrosine kinase coding domain (exons 18-21),wherein deletion in exon 19 (746-753) accounts for about 45% of allmutations, and substitution in exon 21 (mainly L858R) accounts for about40% of all mutations. At present, it is generally believed that thesetwo hot mutations can enhance the sensitivity of tumor cells againstTKI, and can be used as an effective index to predict TKI treatment.Therefore, the detection of EGFR gene mutations has an importantreference value for guiding clinical administration in patients withNSCLC. Methods for detecting EGFR currently used in clinical treatmentinclude: 1) traditional sequencing. This method has high accuracy.However, high requirement on the sample source, long sequencing time,the need for sequence analysis, and high cost of this method limit itsuse in clinic. 2) Polymerase chain reaction-single strand conformationpolymorphism (PCR-SSCP). It is a classical method for detecting genemutations, and can detect unknown mutations. It has advantages of simpleoperation and high sensitivity. However, it also has an obviousdisadvantage, which is the requirement for parallel standard control.Additionally, this method has a high false positive: the detection rateis 75-95% when the tested PCR product is less than 200 bp, while thedetection rate is only about 50% when the tested PCR sample is more than400 bp. 3) Mutation enriched PCR: a two-step PCR using a restrictionendonuclease to selectively digest the wild type EGFR gene. After thefirst PCR, the wild type EGFR is digested selectively and the mutatedEGFR genes are enriched, and then the second PCR is conducted. The PCRproduct is detected by electrophoresis, and whether EGFR is mutated isdetermined based on the detection results of PCR product. This method ishighly sensitive, and can detect one mutation among 10³-10⁴ wild typeEGFRs. However, this method needs twice PCR and enzyme digestion, thusis complex and time-consuming. In addition, there are technologies likeAMRS and micro digital PCR, but their application in clinic still needstime.

Thus, there is an urgent need for a fast and efficient method fordetecting EGFR gene mutations clinically. The inventor found, during theresearch of fragment DNA detection, a new method for detecting DNAfragments, including cyclizing and then amplifying DNA fragments. Basedon this discovery and in combination with the second generation highthroughput sequencing technology, the inventor improved theaforementioned method and designed optimized primers specifically basedon the EGFR genes, and developed a method and a kit thereof forsequencing and analyzing EGFR gene mutations in plasma DNAs.

SUMMARY OF INVENTION

In one aspect, the present invention provides a method fornon-invasively detecting EGFR gene mutations in subjects, comprising thefollowing steps:

-   designing primers according to EGFR gene exons;-   extracting plasma DNAs from the subjects;-   connecting the extracted plasma DNAs with tagging linkers;-   PCR pre-amplifying the tagging linkers connected plasma DNAs;-   cyclizing the amplified DNAs to obtain cyclised DNAs;-   PCR amplifying the cyclised DNAs using the designed primers; and-   high throughput sequencing the PCR amplified product and analyzing    the EGFR gene mutations.

Further, the primers are a pair of primers that are adjacent andbackward extended.

Further, primers of the backward extended primer pair are located on 5′or 3′ end of the EGFR gene exons.

Further, space of the backward extended primer pair is 0-½ of the totalbase pairs of the fragment DNAs.

Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes.

Further, 5′ end of the backward extended primers contains linkersequences for high throughput sequencing library.

Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes, and sequences of the primers are asfollows:

E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.

Further, the cyclization is a splint mediated single strand DNAcyclization.

Further, the EGFR genes in plasma DNAs have insertion, deletion,substitution or gene fusion mutations.

Further, the high throughput sequencing technologies are selected fromRoche/454 FLX, Illumina/Hiseq/Miseq, Applied Biosystems SOLID and lifeTechnologies/Ion Torrent/Proton.

In another aspect, the present invention provides a kit fornon-invasively detecting EGFR gene mutations, comprising: reagents forextracting plasma DNAs, a DNA cyclase, primers and reagents foramplifying target DNAs.

Further, the kit comprises primers and reagents for pre-amplifyingregions to be tested in the EGFR genes.

Further, the kit comprises reagents for high throughput sequencing.

Further, the reagents for high throughput sequencing are applicable tothe following high throughput sequencing technologies: Roche/454 FLX,Illumina/Hiseq/Miseq, Applied Biosystems SOLID and life Technologies/IonTorrent/Proton.

Further, the primers for amplifying regions to be tested in the EGFRgenes are a pair of primers that are adjacent and backward extended.

Further, primers of the backward extended primer pair are located on 5′or 3′ end of the sites or regions to be tested in the EGFR genes.

Further, space of the backward extended primer pair is 0-½ of total basepairs of the plasma DNAs.

Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes.

Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes, and sequences of the primers are asfollows:

E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.

Further, the plasma DNA linkers contain tagging sequences.

Further, the plasma DNAs are pre-amplified before they are cyclised.

Further, the cyclization is a splint mediated single strand DNAcyclization.

Further, the sites or regions to be tested in the EGFR genes haveinsertion, deletion, substitution or gene fusion mutations.

In yet another aspect, the present invention provides a use of primersagainst EGFR gene exons in the preparation of diagnosing reagents orkits for non-invasively detecting EGFR gene mutations in subjects,characterized in that the diagnosing reagents or kits are applicable toa method for non-invasively detecting EGFR gene mutations in subjectscomprising the following steps:

-   extracting plasma DNAs from the subjects;-   connecting the extracted plasma DNAs with tagging linkers;-   PCR pre-amplifying the tagging linkers connected plasma DNAs;-   cyclizing the amplified DNAs to obtain cyclised DNAs;-   PCR amplifying the cyclised DNAs using the designed primers; and-   high throughput sequencing the PCR amplified product and analyzing    the EGFR gene mutations.

Further, the primers are a pair of primers that are adjacent andbackward extended.

Further, primers of the backward extended primer pair are located on 5′or 3′ end of the EGFR gene exons.

Further, space of the backward extended primer pair is 0-½ of total basepairs of the fragment DNAs.

Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes.

Further, 5′ end of the backward extended primers contains linkersequences for high throughput sequencing library.

Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes, and sequences of the primers are asfollows:

E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.

Further, the cyclization is a splint mediated single strand DNAcyclization.

Further, the EGFR genes in the plasma DNAs have insertion, deletion,substitution or gene fusion mutations.

Further, the high throughput sequencing is selected from Roche/454 FLX,Illumina/Hiseq/Miseq, Applied Biosystems SOLID and life Technologies/IonTorrent/Proton.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows experimental design of the present invention. The plasmaDNA is first pre-amplified to increase yield of the detection product,and then the pre-amplified DNA is cyclised to increase availabletemplates for detection. The EGFR gene is amplified to prepare a libraryfor high throughput sequencing.

FIG. 2 shows design principle of the cyclization of the presentinvention. The pre-amplified DNA is a double strand DNA with universallinkers on both ends. Splint with reverse complimentary sequences isdesigned based on the sequences of the universal linkers on both ends,and a partial double strand is formed after annealing and hybridization.Single strand cyclised product is finally obtained by ligation using Tapligase. FIG. 2A shows a general scheme, and FIG. 2B shows thecyclization principle.

FIG. 3 shows cyclization rate of the present invention. This figureshows the imaging results of 6% denaturing gel. 1: a PCR product beforecyclization; 2: a product after cyclization (not digested byexonuclease), M: a marker; 3: an exonuclease digested product aftercyclization (not purified); 4: an exonuclease digested and purifiedproduct after cyclization.

FIG. 4 shows the final EGFR sample library. This figure shows theresults of 2% agarose gel. 1: LC2; 2: LC3; 3: LC113; 4: LC314; 5: LC320;6: NTC(a negative control). The sample is a Lung Cancer (LC) sample.

EMBODIMENTS

With improvements in the sequencing technology, the traditional Sangersequencing has been unable to fully meet the requirement of research.Thus, the second generation sequencing technology with lower cost,higher throughput, faster speed, and having capability for sequencingthe whole genome emerges. The main principle of the second generationsequencing technology is high throughput sequencing by synthesis,namely, determining DNA sequences by capturing newly synthesized endlabelling. The available technologic platforms mainly include Roche/454FLX, Illumina/Genome Analyzer/Hiseq/Miseq, Applied Biosystems SOLID,life Technologies/Ion Torrent, and the like. Taking Illumina product asan example, HiSeq 2000 can reach a sequencing throughput of 30× coveragein 6 human genomes per run, i.e., about 600 G/run, and the operationtime is reduced to 30 minutes. Furthermore, with the maturity of thesecond generation sequencing technology, the investigation on itsclinical application has developed quickly. Research shows that fetusgenetic health can be judged by sequencing maternal plasma DNAs, andthat sequencing plasma DNAs in subjects can be used for early cancerscreening, which will have a wide application in the future.

Plasma DNAs, also known as circulating DNAs, are extracellular DNAs inthe blood and is tens to hundreds of nucleotides in length (a main peakof about 167 bp). It presents in a form of DNA-protein complex, or asfree DNA fragments. Normally, plasma DNAs are derived from DNA releaseof a small amount of senescent and dead cells. Under healthy condition,the generation and removal of the circulating DNAs are in a dynamicequilibrium and are maintained at a relatively steady low level. 1 mLplasma from a normal person contains about 2000 genomic DNAs. Thecirculating DNAs can reflect the metabolic condition of cells in humanbody, thus is an important index for judging health. The change ofquantity and quality of circulating DNAs in peripheral blood is closelyrelated to several diseases (including tumour, complex severe traumata,organ transplantation, pregnancy-related diseases, infectious diseases,organ failure, and the like). As a non-invasive detection index, it isexpected to be an important molecular marker for early diagnosing somediseases, monitoring the conditions, and evaluating the therapeuticeffects and prognosis of the diseases. For example, researches show thatEGFR regulates the cell cycle progress, repair and survival of tumorcells, and at the same time relates to tumor metastasis. Recently,molecular targeted therapy using EGFR as the therapy target has receivedwidespread attention from cancer communities both at home and abroad,and an EGFR tyrosine kinase inhibitor, Iressa (Genfitinib), has beenapproved by America Food and Drug Administration (FDA) for treatingadvanced NSCLC. The prominent feature of molecular targeted drugs isthat its therapeutic effect strongly depends on the target: thetherapeutic effect is significant strong in patients with the “target”,while the therapeutic effect is weak or none in patients without the“target”, which thus delays other treatments and makes the conditionsworse. Therefore, blind administrations without target detection notonly may result in high economic loss, but also may delay the valuabletiming for treatment, or even aggravate the conditions. It is curtail tojudge quickly and accurately whether the patient has the specific targetfor the targeted drug treatment. The traditional EGFR detection mainlydetects the lesion tissue section by FISHor qPCR. However, it is foundthat there are more free DNAs in the plasma of NSCLC patients, about 10times of those of normal people. A large quantity of free DNAs in plasmaderives from DNAs release of senescent and dead tumor cells. They aresimilar to tumor genomic DNAs in genetic characteristics, and mutationsthereof include deletion, point mutation, and increased copy number.EGFR gene mutations can be detected by examining plasma DNAs in NSCLCpatients, and thus make it possible to detect EGFR expressionnon-invasively. The present invention detects EGFR expressions andmutations in plasma DNAs by the second generation sequencing technologyquickly, accurately, non-invasively and with high sensitivity, and thusprovides various diagnosis basis for patients.

In view of the clinical significance of non-invasive detection by plasmaDNAs sequencing and the rapid development of the second generation highthroughput sequencing, the inventor found that sequencing plasma DNAs inlarge-scale can detect EGFR gene expressions and mutations more quickly,accurately, and non-invasively. It is applicable to a variety of secondgeneration high throughput sequencing platforms, including but notlimited to, Roche/454 FLX, Illumina/Genome Analyzer/Hiseq/Miseq, AppliedBiosystems SOLID, life Technologies/Ion Torrent, and the like.

The present invention is based on the following two facts: 1) plasmafree DNAs in patients are similar to genomic DNAs in geneticcharacteristics. The plasma free DNAs of patients are higher in contentthan those of normal people, and often contain lots of mutations, whileeach mutation may be of low frequency; 2)The second generation highthroughput sequencing can obtain the information of plasma free DNAsquickly, accurately, and with high throughput. Combining these two factsenables the non-invasive large scale application of the detection ingenome specific regions. Researches show that plasma DNAs exist asfragments with low amount (1 mL plasma contains about 2000 genomes) andshort length (mainly about 167 bp), which make it difficult for thetraditional PCR to enrich mutations effectively using plasma DNAs astemplates, resulting in rapid decrease in detection sensitivity. Thepresent invention differs from the traditional methods in that the DNAfragments connected with sequence tagging linkers are amplified andsingle strand cyclized, then by means of back-to-back primeramplification, the templates are used maximally and the library issequenced by high throughput paired-end sequencing. The originalamplified templates are assembled based on the original sequencinganalysis, and the tagging sequences are recorded. Sequences with thesame position on the genome and the same tagging sequences arecalculated as one template. The number of templates amplified by everyprimer pair is calculated and the number of mutated template is countedand recorded. The present invention improves the cyclization method andoptimizes primers for the EGFR genes. The unique design of taggingsequences reduces background and prevents contamination effectively. Theunique template set is counted by restoring templates in systemsaccurately, and thus a single molecule detection with high accuracy isfinally achieved.

According to one specific embodiment of the present invention, itprovides a method for non-invasively detecting EGFR gene mutations insubjects, comprising the following steps: (1) designing primersaccording to EGFR gene exons; (2) extracting plasma DNAs from thesubjects; (3) connecting the extracted plasma DNAs with tagging linkers;(4) PCR pre-amplifying the tagging linkers connected plasma DNA; (5)cyclizing the amplified DNAs to obtain cyclised DNAs; (6) PCR amplifyingthe cyclised DNAs using the designed primers; and (7) high throughputsequencing the PCR amplified product and analyzing the EGFR genemutations. “Non-invasive detection” in the present invention means thatin comparison with that routine histology detection methods such assurgery, tissue biopsy and the like, which are directly against cancertissues, will result in body damages in subjects, the present inventiononly detects blood sample from the subjects. Traditional methods fordetecting DNAs or gene fragments require PCR amplification of theregions to be tested before detection, and thus the DNAs or genefragments to be tested should be complete. However, most of the plasmaDNA fragments are incomplete, and thus the DNA fragments that can beused as templates in PCR amplification is few in number and is difficultto be detected by a routine PCR. Therefore, PCR amplification in thepresent invention adopts DNA cycllization technology to transformfragment DNAs into cyclic DNAs using linker sequences and enzymes.Primers based on the regions to be tested are designed; sequencinglibrary is amplified and constructed, and then is sequenced by a highthroughput sequencing technology; and the EGFR gene mutations areanalyzed. FIG. 1 shows principle of the present invention. Plasma DNA isfirst extracted and connected with tagging linkers, which is in Y-shape.The double strand region contains tagging sequences that aredistinguishable between different templates. To increase the amount ofamplificable DNA fragments, pre-amplification can be conducted,universal sequence on the primers and linkers can be annealed, and thenthe pre-amplified DNAs are cyclised, for example, using a splint. FIG. 2shows DNA cyclization principle of the present invention. Splintsequence and the pre-amplified primer region are complementary. Primersaccording to EGFR exons, for example, exon 18, 19, 20 and 21, aredesigned, and specific PCR amplification according to EGFR genes isconducted to generate a sequencing library, which is then sequenced bythe current high throughput sequencing technology to obtain the specificregions in the tested EGFR genes, namely, gene sequences of the exons,and then gene mutations in the specific region are analyzed. The highthroughput sequencing of the present invention refers to the availablesecond generation sequencing techonology, such as Roche/454 FLX,Illumina/Genome Analyzer/Hiseq/Miseq, Applied Biosystems SOLID, lifeTechnologies/Ion Torrent and the like. Different platforms differ insequencing principles. Taking IlluminaMiseq as an example, the generatedsequencing library is denatured and high throughput sequenced in a Miseqsequencer. The read length is 250 bp on both ends, and every primer siteproduces about 100, 000 sequences. The double-end sequences are combinedto a single end sequence based on terminal repeated regions. Linkerregions in the single end sequence are searched, and the sequence isrestored to the original plasma DNA fragments, while the taggingsequence in every sequence is recorded. The restored DNA fragments arecompared with human genome, with the start and terminal coordinates ofthe sequence on the genome recorded. A unique template sequence isobtained after redundancy removal using the start and terminalcoordinates and tagging sequences as standards. The type and ratio ofmutations are determined by counting the template sequences in thetarget zone.

According to another further specific embodiment of the presentinvention, primers of the backward extended primer pair are located on5′ or 3′ end of the EGFR gene exons. Further, space of the backwardextended primer pair is 0-½ of total base pairs of the fragment DNAs.Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes. Further, 5′ end of the backward extendedprimers contains linker sequences for high throughput sequencinglibrary. Further, the backward extended primers aim at exon 18, exon 19,exon 20, or exon 21 of EGFR genes, and sequences of the primers are asfollows:

E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.

Further, the cyclization is a splint mediated single strand DNAcyclization.

Further, the high throughput sequencing technologies are selected fromRoche/454 FLX, Illumina/Hiseq/Miseq, Applied Biosystems SOLID and lifeTechnologies/Ion Torrent/Proton. Illumina technology is used in thepresent invention.

According to another specific embodiment of the present invention, itprovides a kit for non-invasively detecting EGFR gene mutations,comprising: reagents for extracting plasma DNAs, a DNA cyclase, primersand reagents for amplifying target DNAs. Conventional reagents orcommercially available kits can be used in the extraction of plasmaDNAs. Further, the amplification primers of the regions to be tested inthe EGFR genes are a pair of primers that are adjacent and backwardextended. Further, primers of the backward extended primer pair arelocated on 5′ or 3′ end of the sites or regions to be tested in the EGFRgenes. Further, space of the backward extended primer pair is 0-½oftotal base pairs of the plasma DNAs. Further, the backward extendedprimers aim at exon 18, exon 19, exon 20, or exon 21 of the EGFR genes.Further, the backward extended primers aim at exon 18, exon 19, exon 20,or exon 21 of the EGFR genes, and sequences of the primers are asfollows:

E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.

Further, the kit comprises primers and reagents for pre-amplifying theregions to be tested in the EGFR genes. Specifically, the reagents andprimers for pre-amplification include Taq DNA polymerase and its buffer,and primers for pre-amplification that are complementary to the Y-shapelinkers.

Further, the kit comprises reagents for high throughput sequencing.Further, the reagents for high throughput sequencing are applicable tothe following high throughput sequencing technologies: Roche/454 FLX,Illumina/Hiseq/Miseq, Applied Biosystems SOLID and life Technologies/IonTorrent/Proton. Further, the plasma DNA connection linkers containtagging sequences. Further, the plasma DNAs are pre-amplified beforethey are cyclised. Further, the cyclization is a splint mediated singlestrand DNA cyclization.

According to yet another specific embodiment of the present invention,it provides a use of primers according to EGFR gene exons in thepreparation of diagnosing reagents or kits for non-invasively detectingEGFR gene mutations in subjects, characterized in that the diagnosingreagents or kits are applicable to a method for non-invasively detectingEGFR gene mutations in subjects comprising the following steps:

-   extracting plasma DNAs in subjects;-   connecting the extracted plasma DNAs with tagging linkers;-   PCR pre-amplifying the tagging linkers connected plasma DNAs;-   cyclizing the amplified DNAs to obtain cyclised DNAs;-   PCR amplifying the cyclised DNAs using the designed primers; and-   high throughput sequencing the PCR amplified product and analyzing    the EGFR gene mutations.

Further, the primers are a pair of primers that are adjacent andbackward extended. Further, primers of the backward extended primer pairare located on 5′ or 3′ end of the EGFR gene exons. Further, space ofthe backward extended primer pair is 0-½ of total base pairs of thefragment DNAs. Further, the backward extended primers aim at exon 18,exon 19, exon 20, or exon 21 of the EGFR genes. Further, 5′ end of thebackward extended primers contains linker sequences for high throughputsequencing library. Further, the backward extended primers aim at exon18, exon 19, exon 20, or exon 21 of the EGFR genes, and sequences of theprimers are as follows:

E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.

Further, the cyclization is a splint mediated single strand DNAcyclization. Further, the high throughput sequencing technologies areselected from Roche/454 FLX, Illumina/Hiseq/Miseq, Applied BiosystemsSOLID and life Technologies/Ion Torrent/Proton.

EXAMPLES Example 1

The plasma DNA template was amplified using self-designed linkers andaccording to a method for constructing a plasma DNA high throughputsequencing library (that is, PCR pre-amplification using phosphorylatedprimers after linker connection). The PCR product was purified by a gelcutting and cyclised by a splint connection. The cyclised product wasdigested by Exo III, purified, and screened by multiplex PCR with 8pairs of back-to-back primers (the primers contain universal sequencesfor constructing a sequencing library). The mutation sites should beclose to the forward primer or the reverse primer. The library wasfinally obtained by purifying the amplification product amplified by theuniversal primers.

1. Linker design. It is annealed to a double strand, wherein X is atagging sequence:

ssCycADT-1: GTCTCATCCCTGCGTGXXXXT ssCycADT-2: pXXXXCACGCAGGGTACGTGT

The structure of connection product:

Top: GTCTCATCCCTGCGTGXXXXTNNN AXXXXCACGCAGGGTACGTGT Bottom:TGTGCATGGGACGCACXXXXA  NNN TXXXXGTGCGTCCCTACTCTG

Primers:

ssCycUniprimer-F: pGTCTCATCCCTGCGTG ssCycUniprimer-R: pACACGTACCCTGCGTG

The library structure after pre-amplification:

pGTCTCATCCCTGCGTGXXXXTNNN AXXXXCACGCAGGGTACGTGT CAGAGTAGGGACGCACXXXXA NNN TXXXXGTGCGTCCCATGCACAp

Back-to-back primers for amplification in target zones:

EXON18 (123 bp)CAAGTGCCGTGTCCTGGCACCCAAGCCCATGCCGTGGCTGCTGGTCCCCCTGCTGGGCCATGTCTGGCACTGCTTTCCAGCATGGTGAGGGCTGAGGTGACCCTTGTCTCTGTGTTCTTGTCCCCCCCAGCTTGTGGAGCCTCTTACACCCAGTGGAGAAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCCGGTGCGTTCGGCACGGTGTATAAGGTAAGGTCCCTGGCACAGGCCTCTGGGCTGGGCCGCAGGGCCTCTCATGGTCTGGTGGGGAGCCCAGAGTCCTTGCAAGCTGTATATTTCCATCATCTACTTTACTCTTTGTTTCACTGAGTGTTTGG E18-1-3F: CCCAGCTTGTGGAGCCTCE18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:  GCAGGGCCTCTCATGGTCE18-2-R:  CCTGTGCCAGGGACCTTAC EXON19 (99 bp)GCAATATCAGCCTTAGGTGCGGCTCCACAGCCCCAGTGTCCCTCACCTTCGGGGTGCATCGCTGGTAACATCCACCCAGATCACTGGGCAGCATGTGGCACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCACCTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTTTCTATG E19-1-F: ACGTCTTCCTTCTCTCTCTGTCATE19-1-R: GTGAGATGGTGCCACATGCT E19-2-F: GTCCATGGCTCTGAACCTCAE19-2-R: CCACACAGCAAAGCAGAAAC EXON20 (186 bp)CCATGAGTACGTATTTTGAAACTCAAGATCGCATTCATGCGTCTTCACCTGGAAGGGGTCCATGTGCCCCTCCTTCTGGCCACCATGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAGGTAATCAGGGAAGGGAGATACGGGGAGGGGAGATAAGGAGCCAGGATCCTCACATGCGGTCTGCGCTCCTGGGATAGCAAGAGTTTGCCATGGGGATATGE20-1-F:  CACACTGACGTGCCTCTCC E20-1-R:  CTTCGCATGGTGGCAGAE20-2-1F: CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGATEXON21 (156 bp)CTAACGTTCGCCAGCCATAAGTCCTCGACGTGGAGAGGCTCAGAGCCTGGCATGAACATGACCCTGAATTCGGATGCAGAGCTTCTTCCCATGATGATCTGTCCCTCACAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCCTTCCCACTAGCTGTATTGTTTAACACATGCAGGGGAGGATGCTCTCCAGACATTCTGGGTGAGCTCGCAGCE21-1-F: AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGGE21-2-F: TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT

2. Plasma free DNAs were extracted from 2 mL plasma.

3. End-filling:

The reaction mixture was prepared as follows:

TABLE 1 PlasmaDNA solution 38.5 μl T4 DNA phosphorylation buffer (10X) 5μl 10 mM dNTP mixture 2 μl T4 DNApolymerase 2 μl T4 DNA phosphorylase 2μl Klenow enzyme 0.5 μl SterileH₂O 0 μl Total volume 50 μl

The mixture was placed in a 20° C. warm bath for 30 min. The DNA samplewas purified on a purification column and eluted by 42 μl sterile dH₂Oor an elution buffer.

4. Adding poly-adenine tail on the 3′ end of the DNA fragments:

The reaction mixture was prepared as follows:

TABLE 2 End-filled DNA 32 μl Klenow reaction buffer (10X) 5 μl dATPsolution 10 μl klenow ex-enzyme (lacking 3′-5′ exonuclease activity) 3μl SterileH₂O 0 μl Total volume 50 μl

The mixture was placed in a 37° C. warm bath for 30 min. The DNA samplewas purified on a column and eluted by 25 μl sterile dH₂O or an elutionbuffer.

5. Connecting linkers to the DNA fragments

The reaction mixture was prepared as follows:

TABLE 3 End-filled dA-tailed DNA 33 μl Reaction buffer for quickconnection (5X) 10 μl 5 μm DNA linker 2 μl Quick T4 DNA ligase (NEB) 5μl Total volume 50 μl

The mixture was placed in a 20° C. warm bath for 15 min. The DNA samplewas purified on a Qiagen column and eluted by 25 μl sterile dH₂O or anelution buffer.

6. Enriching the linker-modified DNA fragments by PCR pre-amplification

The PCR reaction mixture was prepared as follows:

Buffers EB 14 uL 10X Taq ligase buffer 5 uL Split Oligo (10 μM) 4 uLPre-lib 25 uL Taq ligase 2 uL Total volume 50 uL

PCR programs:

95° C. 30 s 30 cycles 50° C. 2 min  4° C. stop

TABLE 4 DNA 12.5 μl Phusion DNA polymerase (Phusion DNA polymerase 25 μlmixture) PCR primer mixture 2 μl Ultrapure water 10.5 μl Total volume 50μl

Amplification using the following PCR programs:

a. 98° C. 30 s;

b. 18 cycles as follows:

98° C. 10s, 65° C. 30 s, 72° C. 30 s;

c. 72° C. 5 min;

d. maintained at 4° C.

6. PCR product was analyzed by electrophoresis on 2% agarose gel, andthe results were shown in FIG. 4. The gel with target band of 200 bp wascut and recycled using a Qiagen Kit (FIG. 4), and eluted by 20 μlelution buffer.

7. Cyclization

The cyclization system was prepared as follows (Table 5)

DNA template 12 ul Circ Ligase II 10X reaction buffer 2 ul 50 mM MnCl₂ 1ul 5M Betaine (optional): 4 ul Circ Ligase II ssDNA ligase (100 U) 1 ulTotal volume 20 ul

Reaction conditions

60° C. 1 h 80° C. 10 min  4° C. stop

8. Enzyme digestion:

All the cyclization products were digested by Exo III, and the digestionsystem was

10 × NE buffer 1.2 ul ExoIII 1 ul Cyclization product 10 ul

The digestion system was placed in a PCR machine, and reacted for 30 minat 37° C.

The digested product was purified on a purification column and dissolvedin 30 ul EB buffer. The concentration was measured by Qubit, and theresults were as follows:

Sample Number Concentration 2 4 ng/ul 3 0.3 ng/ul 4 2.33 ng/ul 5 5.74ng/ul

Examples of the present invention used a splint-mediated cyclization,which has a high rate of cyclization. Detailed tracking and detectingresults of every step during the cyclization process were shown in FIG.3. Lane 3 shows the cyclization product after digestion by exonuclease,which is the target product. The cyclization rate was about 20%.

9. Reverse PCR screening of the target zone using back-to-back primers

PCR reaction system was prepared as follows

TABLE 6 dd H₂O 13 or 18 ul AmpliTaq Gold ® 360 Master Mix (2×) 25 ulCycEGFR18-F1 1 ul CycEGFR18-R1 1 ul DNA 10 or 5 ul Total volume 50 ulNote: the controls were P (with primers and without template) and N(template is un-cyclized ssCyc Lib)

PCR reaction conditions

TABLE 7 95° C. 10 min 1 cycle 95° C. 30 s 30 cycles 55° C. 30 s 72° C.30 s 72° C. 5 min 1 cycle

10. The second round PCR

The second round PCR was performed using products of the reverse PCR astemplates. System

TABLE 8 Phusion PCR Master Mix (2×) 25 uL P5-B1-F (10 mM) 1 uL Primer 2-index 1-2 (10 uM) 1 uL Products of reverse PCR 5 uL ddH₂O add to 50 uL

Programs:

98° C. 30 s 1 cycle 98° C. 30 s 12 cycles 65° C. 30 s 72° C. 30 s 72° C.5 min 1ycle

10 uL PCR product from the second round PCR was analyzed byelectrophoresis on 2% agarose gel, and the results were shown in FIG. 4.

11. The remaining 40 uL PCR product from the second round PCR waspurified on a QIAGEN column, and dissolved in 20 uL EB buffer togenerate the final library.

12. After quality control, the generated library was 250 bp double-endsequenced by IlluminaMiseq.

13. Every high throughput sequenced double-end sequences were assembledto one sequence based on repeated regions. Linkers were removed, and thesequence was restored to the original template sequence, which was thencompared to the human genome (hg19). The unique template sequence setwas counted by comparing the start and terminal coordinates and taggingsequences of the template sequence on the genome. Using the uniquetemplate sequence, genome coverage was then calculated, which can beused for evaluating the specificity of the library and calculating thesomatic cell mutations in the EGFR region.

The results were compared with an EGFR gene mutation detection kit fromAmoyDx (directed to the same cancer tissue) and a digital PCR. Thecomparison results were:

ARMS ddPCR Results Results (positive oil Sequencing Results Sample(AmoyDx droplets/total (positive templates/ Number kit) oil droplets)total templates) LC113 19-del 19-del (2/723) 19-del c.2239_2251>C(7/1192) LC314 WT WT (del: 0/34; WT (0/876) 1858r: 0/178; t790: 0/208)LC320 19-del 19-del (1/161) 19-del c.2236_2250del15 (9/909) LC2 WT n/aWT (0/668) LC3 L858R L858R (589/858) c.2573T>G; p.L858R (1599/2510)

5 samples tested by the method of the present invention showed highlyconsistent results with those tested by other methods. AMRS-PCR(amplification refractory mutation system) Taq DNA polymerase lacks3′-5′ exonuclease activity. Under certain conditions, effectiveamplification occurs only when the last base on 3′ end of PCR primers iscomplementary to the template DNA. Mutated genes and wild type genes canbe directly distinguished by PCR using suitable primers directed todifferent known mutations. This method is mainly for biopsy and FFPEsamples. 5 mL peripheral blood before surgery and FFPE samples aftersurgery from the same patient were provided. The mutation type of FFPEsamples on the known sites was detected by ARMS-PCR, and then plasma DNAwas detected by digital PCR and the method of the present invention,respectively.

Example 2

Reliability of the present method was verified by detecting known cancermutation sites. The method was the same as that disclosed in Example 1.

Cancer cell line DNA: cell DNAs containing hybrid c.2235_2249de115 (exon19) mutation were broken by ultrasonication, 166±10 bp fragments wererecycled and mixed with plasma DNAs from a normal person at certainratio. The sensitivity and stability of the method were examined.

The detection results of c.2235_2249del15 were as follows:

Nos. of Nos. of Nos. of Sample Total Non-Del Del Nos. Predictiontemplates templates templates ratio Sample 1 0% mutation 862 862 0 0.00%Sample 2 0.1 mutation 438 433 5 1.09% Sample 3 1% mutation 905 843 626.78% Sample 4 5% mutation 880 786 94 10.67% Sample 5 25% mutation 16311017 614 37.65%

What is claimed is:
 1. A method for non-invasively detecting EGFR genemutations in subjects, comprising the following steps: designing primersaccording to EGFR gene exons; extracting plasma DNAs from the subjects;connecting the extracted plasma DNAs with tagging linkers; PCRpre-amplifying the tagging linkers connected plasma DNAs; cyclising thepre-amplified DNAs to obtain cyclised DNAs; PCR amplifying the cyclisedDNAs using the designed primers; and high throughput sequencing the PCRamplified product and analyzing the EGFR gene mutations.
 2. The methodaccording to claim 1, characterized in that the primers are a pair ofprimers that are adjacent and backward extended.
 3. The method accordingto claim 2, characterized in that primers of the backward extendedprimer pair are located on 5′ or 3′ end of the EGFR gene exons.
 4. Themethod according to claim 2, characterized in that space of the backwardextended primer pair is 0-½ of total base pairs of the DNAs.
 5. Themethod according to claim 3, characterized in that the backward extendedprimers aim at exon 18, exon 19, exon 20, or exon 21 of the EGFR genes.6. The method according to claim 3, characterized in that 5′ end of thebackward extended primers contains linker sequences for high throughputsequencing library.
 7. The method according to claim 3, characterized inthat the backward extended primers aim at exon 18, exon 19, exon 20, orexon 21 of the EGFR genes, and sequences of the primers are as follows:E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.


8. The method according to claim 1, characterized in that thecyclization is a splint mediated single strand DNA cyclization.
 9. Themethod according to claim 1, characterized in that the EGFR genes inplasma DNAs have insertion, deletion, substitution or gene fusionmutations.
 10. The method according to claim 1, characterized in thatthe high throughput sequencing technologies are selected from Roche/454FLX, Illumina/Hiseq/Miseq, Applied Biosystems SOLID and lifeTechnologies/Ion Torrent/Proton.
 11. A kit for non-invasively detectingEGFR gene mutations, comprising: reagents for extracting plasma DNAs, aDNA cyclase, primers and reagents for amplifying target DNAs.
 12. Thekit according to claim 11 for non-invasively detecting EGFR genemutations, further comprising primers and reagents for pre-amplifyingregions to be tested in the EGFR genes.
 13. The kit according to claim11 for non-invasively detecting EGFR gene mutations, further comprisingreagents for high throughput sequencing.
 14. The kit according to claim13 for non-invasively detecting EGFR gene mutations, characterized inthat the reagents for high throughput sequencing are applicable to thefollowing high throughput sequencing technologies: Roche/454 FLX,Illumina/Hiseq/Miseq, Applied Biosystems SOLID and life Technologies/IonTorrent/Proton.
 15. The kit according to claim 11 for non-invasivelydetecting EGFR gene mutations, characterized in that the primers foramplifying target DNAs are a pair of primers that are adjacent andbackward extended.
 16. The kit according to claim 15 for non-invasivelydetecting EGFR gene mutations, characterized in that primers of thebackward extended primer pair are located on 5′ or 3′ end of the sitesor regions to be tested in the EGFR genes.
 17. The kit according toclaim 15 for non-invasively detecting EGFR gene mutations, characterizedin that space of the backward extended primer pair is 0-½ of total basepairs of the plasma DNAs.
 18. The kit according to claim 15 fornon-invasively detecting EGFR gene mutations, characterized in that thebackward extended primers aim at exon 18, exon 19, exon 20, or exon 21of the EGFR genes.
 19. The kit according to claim 17 for non-invasivelydetecting EGFR gene mutations, characterized in that the backwardextended primers aim at exon 18, exon 19, exon 20, or exon 21 of theEGFR genes, and sequences of the primers are as follows: E18-1-3F:CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.


20. The kit according to claim 15 for non-invasively detecting EGFR genemutations, characterized in that 5′ end of the backward extended primerscontains linker sequences for high throughput sequencing library. 21.The kit according to claim 20 for non-invasively detecting EGFR genemutations, characterized in that the linker sequences contain taggingsequences.
 22. The kit according to claim 11 for non-invasivelydetecting EGFR gene mutations, characterized in that the plasma DNAs arepre-amplified before they are cyclised.
 23. The kit according to claim22 for non-invasively detecting EGFR gene mutations, characterized inthat the cyclization is a splint mediated single strand DNA cyclization.24. The kit according to claim 12 for non-invasively detecting EGFR genemutations, characterized in that regions to be tested in the EGFR geneshave insertion, deletion, substitution or gene fusion mutations.
 25. Ause of primers according to EGFR gene exons in the preparation ofdiagnosing reagents or kits for non-invasively detecting EGFR genemutations in subjects, wherein the diagnosing reagents or kits areapplicable to a method for non-invasively detecting EGFR gene mutationsin subjects comprising the following steps: extracting plasma DNAs fromthe subjects; connecting the extracted plasma DNAs with tagging linkers;PCR pre-amplifying the tagging linkers connected plasma DNAs; cyclizingthe pre-amplified DNAs to obtain cyclised DNAs; PCR amplifying thecyclised DNA using primers; and high throughput sequencing the PCRamplified product and analyzing the EGFR gene mutations.
 26. The useaccording to claim 25, characterized in that the primers are a pair ofprimers that are adjacent and backward extended.
 27. The use accordingto claim 25, characterized in that primers of the backward extendedprimer pair are located on 5′ or 3′ end of the EGFR gene exons.
 28. Theuse according to claim 26, characterized in that space of the backwardextended primer pair is 0-½ of total base pairs of the DNAs.
 29. The useaccording to claim 26, characterized in that the backward extendedprimers aim at exon 18, exon 19, exon 20, or exon 21 of the EGFR genes.30. The use according to claim 26, characterized in that 5′ end of thebackward extended primers contains linker sequences for high throughputsequencing library.
 31. The use according to claim 26, characterized inthat the backward extended primers aim at exon 18, exon 19, exon 20, orexon 21 of the EGFR genes, and sequences of the primers are as follows:E18-1-3F: CCCAGCTTGTGGAGCCTC E18-1-3R: GACAAGAACACAGAGACAAGGGT E18-2-F:GCAGGGCCTCTCATGGTC E18-2-R: CCTGTGCCAGGGACCTTAC E19-1-F:ACGTCTTCCTTCTCTCTCTGTCAT E19-1-R: GTGAGATGGTGCCACATGCT E19-2-F:GTCCATGGCTCTGAACCTCA E19-2-R: CCACACAGCAAAGCAGAAAC E20-1-F:CACACTGACGTGCCTCTCC E20-1-R: CTTCGCATGGTGGCCAGA E20-2-1F:CCTCCCCGTATCTCCCT E20-2-1R: GGAGATAAGGAGCCAGGAT E21-1-F:AGCAGGGTCTTCTCTGTTTCA E21-1-R: GAGGGACAGATCATCATGGG E21-2-F:TTTCCTGACACCAGGGACC E21-2-R: TGACCTAAAGCCACCTCCTT.


32. The use according to claim 25, characterized in that the cyclizationis a splint mediated single strand DNA cyclization.
 33. The useaccording to claim 25, characterized in that the EGFR genes in plasmaDNAs have insertion, deletion, substitution or gene fusion mutations.34. The use according to claim 25, characterized in that the highthroughput sequencing technologies are selected from Roche/454 FLX,Illumina/Hiseq/Miseq, Applied Biosystems SOLID and life Technologies/IonTorrent/Proton.